Project Summary The evolution of breast cancer involves a complex interplay between the epithelial cells and the surrounding stromal microenvironment. Tumor progression is associated with marked remodeling of the extracellular matrix (ECM), which creates a scaffold that favors invasion and migration. The remodeled ECM is also significantly stiffer than normal, and promotes malignant transformation and metastasis. We hypothesize that ECM stiffness also regulates response to therapy. The ECM provides biochemical and biophysical cues that activate key molecular pathways that direct cell fate and these cues are primarily transduced through the integrin family of receptors. Indeed, ECM tension enhances integrin activity and stimulates PI3-kinase (PI3-k) signaling to induce malignant transition, and ECM stiffness activates the YAP-TAZ transcription factors to enhance the malignant behavior of tumors. As such, PI3-k and YAP-TAZ represent key ECM activated molecular mechanisms that drive the malignant behavior of tissues and could provide novel opportunities for predicting prognosis and therapeutic targeting. Radiation therapy is commonly used to treat breast cancer, however, knowledge of the mechanisms by which stromal tension act to promote resistance is an innovative front of research, and yet uninvestigated. Our preliminary data indicate that high ECM stiffness (4kPa) modifies the apoptotic and proliferative response to ionizing radiation (IR) in malignant breast cells, and reveal that this is associated with YAP activation. Equally important and interesting is the role of tension in therapy resistance in in situ cancer. Ductal carcinoma in situ (DCIS) is comprised of cancerous epithelial cells that reside within the ductal epithelial compartment without yet having invaded into the surrounding stroma. It is well known that active remodeling of the ECM, occurs in the stroma surrounding in situ lesions and that stromal remodeling leads to increased tissue force and collagen-cross-linkingthat drives progression. The architecture of DCIS-like lesions provides unique opportunities to investigate the interplay between cancer cells residing in the duct and stromal activation, and we have developed models of in situ disease for this purpose. We hypothesize that the high tension of an activated ECM stroma plays a critical role in the survival and proliferation of cancerous epithelial cells through direct engagement of the YAP pathways, and that disengaging pro-survival mechanosignaling emanating from the activated stroma is a promising approach towards enhancing therapeutic efficacy. Our goals are to determine if and how ECM tension specifically regulates IR therapy resistance by testing 1) whether the YAP pathways are acting independently in IR- mediated cell death and proliferation; 2) and whether integrin mechanosignaling modifies this regulation. In addition, we have the unique opportunity to investigate whether stromal biophysical and morphological features are associated YAP signaling and disease progression in clinical DCIS.